1. Field of the Invention
The present invention relates to improved recombinant mammalian, particularly human and murine, nucleic acid sequences for granulocyte macrophage-colony stimulating factor (GM-CSF) and their use in improving response to vaccines or gene therapy used for treating or preventing disease.
2. Background of the Invention
Granulocyte macrophage-colony stimulating factor, a soluble secreted glycoprotein, is a potent immunomodulatory cytokine known to facilitate development and prolongation of both humoral and cellular mediated immunity. Additional activities include, for example, activation and enhanced maturation of antigen presenting cells, increasing the expression of MHC class II antigens, activation of mature granulocytes, macrophages and monocytes, and proliferation and differentiation of hematopoietic progenitor cells. These and other diverse immunomodulatory activities have made GM-CSF an attractive investigational cytokine for use as a vaccine adjuvant for improving the immune response to vaccines, including those used for the treatment of cancer and HIV. The cDNA for GM-CSF is known and its expression in cultured cells described previously.
Several strategies have been investigated using GM-CSF as an adjuvant. GM-CSF is a soluble, secreted protein. Vaccine strategies have been described wherein soluble GM-CSF is expressed in vitro (e.g., in yeast, bacteria, or cultured mammalian cells; U.S. Pat. Nos. 5,298,603 and 5,679,356), purified, and administered concomitantly with an immunogen, which may be derived from pathogens (e.g., viruses or bacteria), or molecules associated with cancer, such as melanoma antigen (MAGE). This approach has several problems, including the cost associated with heterologous expression of GM-CSF, non-native glycosylation patterns, and the short half-life observed with systemically administered proteins, which are degraded rapidly or diffuse away from the site of the immunogen.
Fusion proteins which comprise GM-CSF in tandem with a second protein, such as an antigen as described in U.S. Pat. No. 5,108,910, have also been described for use as an adjuvant, however, these compositions suffer the same problems described above with respect to the systemic administration of a purified protein, such as limited half-life, in addition to difficulties arising from unpredictable changes in tertiary structure that may affect the biologic activity of GM-CSF. An alternative approach to administering GM-CSF protein is gene therapy wherein DNA encoding GM-CSF protein is administered and subsequently taken up by the receipient""s cells and GM-CSF protein produced in vivo. This approach offers the advantages of locally sustained release of the cytokine and has proven successful in murine models (Warren T L et al., xe2x80x9cUses of granulocyte-macrophage colonly-stimulating factor in vaccine development,xe2x80x9d Curr. Opin. in Hematology 2000;7:168-173. In addition, recipient cells can be removed from the organism, transfected with GM-CSF DNA, and then reintroduced into the organism at the desired location. The expression levels observed from these approaches may be insufficient for providing the intended immunomodulatory adjuvant activity. Recombinant forms of human GM-CSF have been created with the goal of improving the activity or permitting large scale protein production, however, these sequences alter the amino acid sequence and have been associated with adverse immunologic responses, presumably due to formation of novel epitopes that result from recombinant genes or exposed native protein backbone which is normally blocked by glycosylation (Gribben et al., xe2x80x9cDevelopment of antibodies to unprotected glycosylation sites on recombinant human GM-CSF,xe2x80x9d Lancet 1990 Feb. 24; 335(8687):434-7). For example, sargramostim differs from the native human GM-CSF by one amino acid at position 23 and a different carbohydrate moiety. In contrast, the present invention provides for improved human and murine GM-CSF encoding nucleic acid sequences that encode the identical amino acid sequence as the native gene but with improved in vivo adjuvant activity. The GM-CSF nucleic acid sequences of the present invention alter select nucleotides within the native sequence without altering the amino acid sequence of the native protein.
It is well know that most amino acids are specified by more than one trinucleotide codon and that codon usage varies across species with G+C bias in mammals compared with A+T usage in microorganisms, yeast, and viruses. Selective codon usage has been further characterized by the differences in gene expression observed across different genes from the same species. Genes that are expressed with high frequency include a bias toward particular codons. The codons associated with highly expressed genes also vary with species. The different frequency of codons observed across and within species has been described and categorized by frequency of appearance in the genome (e.g., Nakamura Y., et al., xe2x80x9cCodon usage tabulated from the international DNA sequences databases: status for the year 2000, xe2x80x9dNucl. Acids Res. 28, 292 (2000)). These differences, such as A and T preferences in the third position of a codon observed in bacteria, have been incorporated into human genes, for example, for enhancing expression of heterologous genes in microorganisms (N. D. Connell, xe2x80x9cExpression systems for use in actinomycetes and related organisms,xe2x80x9d Curr. Opin. Biotechnol., 2001, October:12(5):446-9). U.S. Pat. No. 6,288,303 discusses optimizing the codon usage of mammalian genes for optimal expression in plant cells.
As described in detail herein below, the present invention provides for human and murine GM-CSF nucleic acid sequences that comprise codons associated with high expression levels in humans and mice, respectively, and surprisingly result in improved adjuvant activity compared with the native sequence.
The codon optimized mammalian, particularly human and murine GM-CSF nucleic acid sequences of the present invention, although encoding the same amino acid sequence as the native human or murine GM-CSF, provide enhanced adjuvant activity when expressed in vivo compared to the native GM-CSF gene sequence. The present invention provides modified GM-CSF DNA sequences wherein codons in the native gene have been replaced with trinucleotide codons identified in highly expressed genes of the same host (i.e., humans or mice).
The codon optimized GM-CSF sequences of the present invention preserve the native amino acid sequence encoded by the host, and therefore, all native glycosylation sites and processing signals, but result in improved biologic responses when used as a vaccine adjuvant compared to the native gene or recombinant GM-CSF genes of the prior art. The codon optimized GM-CSF nucleic acid sequences are utilized, according to the present invention, for improved biologic responses in support of prevention and treatment of disease by vaccines and gene therapy, and for research purposes.